Single-Molecule Forster Resonance Energy Transfer With a Minimalistic 3D-Printed Setup and Dyes in the Blue-Green...

Förster Resonance Energy Transfer (FRET) is a powerful technique for the detection and characterization of biomolecular interactions and conformational changes with subnanometer spatial resolution and a temporal resolution down to the timescale of fluorescence. While the technique is widely adopted in structural biology and biophysics, the evolution of single-molecule FRET has led to experimental setups with sophisticated optical layouts, multilaser excitation schemes, and time-resolved detection electronics. We here present an accessible alternative toward single-molecule FRET based on Brick-MIC, a recently introduced three-dimensional (3D)-printed microspectroscopy platform. The FRET-Brick uses continuous-wave excitation at 488 nm with a minimal set of optomechanical components and photomultiplier detectors (PMTs). With this, we were able to significantly reduce the setup complexity retaining single-molecule sensitivity with dyes matching the sensitivity of PMTs. To maximize the photon output of Alexa488, ATTO488 (donors), Alexa555, ATTO542, and Cy3B (acceptors), we introduce ferrocene derivatives as photostabilizers that increase both dye brightness and remove dark-states. We benchmark the performance of the FRET-Brick with fluorophore-labeled oligonucleotide reference structures also in comparison to accessible volume simulations, and by detecting conformational changes in bacterial substrate-binding proteins. Our work demonstrates that qualitative and quantitative single-molecule FRTE (smFRET) measurements are possible with the minimalistic and cost-effective FRET-Brick.